Cell conditioned media (CCM) such as macrophage conditioned medium (MCM) is an essential supplement necessary to support the survival and growth of hybridoma cells used in the production of monoclonal antibodies. The use of macrophage cell lines as part of hybridoma technology has proven to be an effective and inexpensive production source of macrophage derived growth factors. Despite the widespread use of MCM as a hybridoma culture supplement there is limited guidance and standardization for MCM production to achieve optimal hybridoma survival and growth. As an undefined supplement, significant variations in production of MCM may negatively impacts hybridoma cell survival and growth.
Monoclonal antibodies (mAbs) remain an essential molecular tool for the selective and specific detection of biological and chemical antigens. A defining property of a mAb is its recognition and binding to a single epitope, a characteristic that has been exploited to achieve exquisite antigen selectivity and discrimination. Hybridoma technology has allowed for the production of mAbs to specific antigens following splenocyte cell fusion from an immunized animal with immortalized myeloma-derived cell lines. The resulting hybridoma cells can be screened for selective mAb production and single cells cloned to yield an immortal hybridoma cell line for continuous production of a desired mAb.
Hybridoma technology has evolved along with modem cell culture practices, benefiting from refined cell media formulations and growth supplements, resulting in improved work flow and consistent mAb production. However, there remains a need for cost effective animal derived culture supplements to support hybridoma cell survival and growth. Indeed, newly formed hybrid cells require the addition of a cell feeder layer (i.e. macrophages) or supplementation with a cell-free conditioned medium for initial stabilization and growth (2, 3). The use of a feeder cell layer (4-6) imposes several disadvantages that include the interference with hybridoma cell growth and the introduction of potential contamination. Therefore, cell-free conditioned medium from continuous cell lines such as fibroblasts (7) or macrophages (8-10) has been commonly used as a culture medium supplement. Conditioned medium collected from the widely available macrophage J774A.1 cell line has been shown to improve hybridoma survival and cloning efficiency after cell fusion (8).
Efforts have been made to identify defined medium conditions to support hybridoma growth and increase the efficiency of antibody production. Proteins such as IL-1, tumor necrosis factor (TNF) (11), granulocyte-macrophage colony-stimulation factor (GM-CSF) (12), and IL-6 (13) have been shown to support hybridoma growth resulting in proprietary blends of define hybridoma growth medium. Although these commercial products can be effective they can be cost prohibitive and many laboratories rely on in-house production of MCM to support hybridoma growth during cell selection and cloning.
Limited guidance and standardization of MCM quality has been reported. This reflects the lack of a defined assay to evaluate the quality of MCM prior to use as a culture supplement. Herein is described optimization of conditioned media designed to optimize MCM production by exploiting a new hybridoma cell line RMH359 that is dependent on J774A.1 derived MCM for survival and growth. Using RMH359 cells we define a novel cellular bioassay for the evaluation of MCM bioactivity in support of hybridoma cell survival and growth. Using the RMH359 bioassay we define optimal conditions for MCM production to achieve maximal bioactivity in support of hybridoma cells and provide a novel method for MCM validation and standardization that ensure hybridoma supplementation with high quality MCM.
The lack of an available method for standardization of MCM bioactivity has limited validation, optimization, and commercial production. Consequently, variations in batch production of MCM may result in low quality MCM that limits hybridoma viability and negatively impacts monoclonal antibody production. Herein is described a novel bioassay using a newly generated MCM-dependent RMH359 hybridoma cell line that can be used to validate MCM bioactivity and standardize production.